Liver engrafting cells, assays, and uses thereof

ABSTRACT

A substantially enriched mammalian hepatic liver engrafting cell population is provided. Methods are provided for the isolation and culture of this liver engrafting cell. The progenitor cells are obtained from a variety of sources, including fetal and adult tissues. The cells are useful in transplantation, for experimental evaluation, and as a source of lineage and cell specific products, including mRNA species useful in identifying genes specifically expressed in these cells, and as targets for the discovery of factors or molecules that can affect them.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/177,178, filedJun. 22, 2002, which claims priority to U.S. Ser. No. 60/300,535, filedJun. 22, 2001. Each of these applications is herein incorporated byreference in its entirety.

The body depends on the liver to perform a number of vital functions,including regulation, synthesis, and secretion of many substancesimportant in maintaining the body's normal state; storage of importantnutrients such as glycogen (glucose), vitamins, and minerals; andpurification, transformation, and clearance of waste products, drugs,and toxins. However, its distinctive characteristics and activitiesrender it susceptible to damage from a variety of sources, and suchdamage can have enormous impact on a person's health.

The most abundant and metabolically active cells in the liver are thehepatocytes. The lobules of the liver are hexagonal in shape, with sixportal triads at the periphery, each containing a branch of the portalvein, a branch of the hepatic artery, and a bile duct, all held tightlytogether by a layer of hepatocytes. Hepatocytes rarely divide, but theyhave a unique capacity to reproduce in response to an appropriatestimulus, such as the removal of a portion of liver. This processinvolves controlled hyperplasia, that usually restores the liver towithin 5 to 10% of its original weight.

The liver has a unique capacity to regenerate after injury. The processbegins with proliferation of “mature” hepatocytes; other cell lineagesincluding biliary epithelial cells (BEC) and sinusoidal cellsproliferate somewhat later. Liver regeneration plays an important roleafter partial hepatectomy and after injuries that destroy portions ofthe liver, such as viral, toxic, or ischemic damage. However, excessivedamage can reach a “point of no return”, and normal tissue is thenreplaced with scar tissue. The liver's ability to regenerate is alsocompromised by pre-existing or repeated liver damage or disease.

It has been found that a number of surface determinants are sharedbetween bone-marrow derived stem cells, and cells that can give rise tohepatocytes, including c-kit, CD34, and Thy-1 in rodents, and c-kit andCD34 in humans (see Oman et al., (1997) Hepatology 26: 720-727; Lemmeret al. (1998) J. Hepatol 29: 450-454; Peterson et al. (1998) Hepatology27: 433-445; ibid (1999) Science 284:1168-1170; Baumann at al., (1999)Hepatology 30: 112-117; Lagasse et al. (2000) Nature Med. 11:1229-1234).These findings may have important clinical implications for gene therapyand hepatocyte transplantation, two innovative approaches to thetreatment of fulminant hepatic failure and genetic metabolic disordersof the liver.

Some evidence has indicated that some immature liver cell lines candifferentiate into both BEC and hepatocytes. For example, Fiorino at al.(1998) In Vitro Cell Dev Biol Anim 34(3):247-58 report isolation of aconditionally transformed liver progenitor cell line. Coleman andPresnell (1996) Hepatology 24(6):1542-6 discuss phenotypic transitionsin proliferating hepatocyte cultures that suggest bipotentdifferentiation capacity of mature hepatocytes. Oval cell precursors arethought to be located either in the canals of Herring or next to thebile ducts. Bile duct cells are required for oval cell proliferation,indicating that either it is the source of the precursors or it acts ina supportive or inductive role. Kubota et al., International PatentApplication WO02/28997 discloses an ICAM-1 expressing progenitor cellpopulation.

Intermediate filament proteins, particularly bile duct-specificcytokeratin 19 (CK19) and the hepatocyte-specific HepParl antigen canhelp define the developmental stages of hepatic progenitor cells duringliver morphogenesis. Ductular hepatocytes proliferate and sharephenotypic characteristics with hepatocytes and BEC. As hepatocytedifferentiation progresses, expression of HepParl antigen increases, andexpression of CK14 and CK19 are lost. In contrast, as progenitor cellsare transformed into ductal plate cells, CK19 expression increases indifferentiated bile ducts, while CK14 and HepParl antigens are lost.Hepatic progenitor cells therefore may differentiate in steps marked bythe acquisition or loss of specific phenotypic characteristics.Commitment of the progenitor cells to either hepatocyte or bile ductepithelial cell lineages results in increased expression of one markerand loss of the other marker. Early reports suggested the in vivopresence of such bipotent progenitor cells may be found in Douarin(1975) Med. Biol. 53:427-455; Shiojiri et al., (1991) Cancer Res. 51:2611-2620; Haruna et al. (1996) Hepatology 23(3):476-81; Tateno andYoshizato (1996) Am J Pathol 149(5):1593-605; and Haque et al. (1996)Lab Invest 75(5):699-705. The expression of albumin andalpha-fetoprotein are also useful markers for hepatocytes.

A discussion of hepatic progenitor cells may be found in Susick at al.(2001) Ann. N.Y. Acad. Sci. 944:398-419.; in U.S. Pat. No. 5,576,207;and U.S. Patent Application no. 20020016000.

To achieve a further characterization of hepatic progenitor cells, andthe cells derived therefrom, it is critical to have well defined modelsystems, that can decipher the complex interplay between “environmental”factors and intrinsic cellular factors that regulate cell renewal, aswell as the phenotypic definition of the specific cells capable ofgiving rise to mature hepatic cells. Identification and characterizationof factors regulating specification and differentiation of cell lineagesin the developing and adult liver, and in the biliary tree are of greatinterest. The further characterization of liver engrafting cells is ofgreat scientific and clinical interest.

SUMMARY OF THE INVENTION

Methods are provided for the separation and characterization of liverengrafting cells (LEC), which are progenitor cells having the ability toengraft the liver and give rise to differentiated hepatic cells. Thecells can be separated on the basis of forward scatter andautofluorescence, and/or by expression of specific cell surface markers.The cells are useful in transplantation, for experimental evaluation,and as a source of lineage and cell specific products, including mRNAspecies useful in identifying genes specifically expressed in thesecells, and as targets for the discovery of factors or molecules that canaffect them.

In vitro and in vivo systems are provided for the growth and analysis,including clonal analysis, of liver engrafting cells. Clonogenic assaysmay be performed in vitro in the presence of a feeder layer of stromalcells. The cells can also be expanded in vitro in the absence of feederlayers. These culture systems are suitable for growth andcharacterization of liver engrafting cells. In vivo the cells engraftthe liver, and engraftment may be experimentally tested by repopulationof liver, cells in FAH deficient animals.

The liver engrafting cells find use in the evaluation of therapiesrelating to liver specific viruses, e.g. hepatitis A, B, C, D, Eviruses, etc., particularly human hepatitis viruses. The cells also finduse in toxicology testing, for the production of hepatocytes in culture,and as a means of providing the by-products of liver metabolism, e.g.the products of drug transformation by liver cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the staining of human fetal liver cells for forwardscatter, autofluorescence, and viability (propidium iodide), andseparation into an R1 and R2 population on the basis of thesecharacteristics. FIG. 1B shows the expression of the 5E12 and HLA ClassI epitopes on subpopulations of cells in the R2 population.

FIGS. 2A and 2B show that the R2 population is heterogeneous forexpression of albumin and CK19, prior to sorting for 5E12 expression.

FIGS. 3A to 3D shows phenotypic analysis of human fetal liver cells.

FIG. 4 shows the staining of cells from the R2 population with 5E12,EpCAM, CD49f, E-Cadherin, and HLA. FIGS. 4A, 4D and 4G show a 5E12 vs.

HLA class I staining. The polygonal regions illustrate the gates used toselect for 5E12⁺, HLA^(low) LEC. FIGS. 4B, 4E and 4H show correspondingplots utilizing E-cadherin; EpCam and CD49f, respectively, as the xaxis. FIGS. 4C, 4F and 41 show the analysis of the populations gated inFIGS. 4B, 4E and 4H, for expression of 5E12. The data demonstrateequivalence of staining between 5E12, EpCam, E-cadherin and CD49f.

FIGS. 5A-5F show staining for albumin (alb), alpha-fetoprotein (afp) andCK19 on colonies derived from human fetal liver LEC after two weeks inculture in vitro.

FIGS. 6A and 6B show the levels of circulating human alpha-1-antitrypsin(AAT)(9A) and albumin (ALB) (9B) protein from serum of NOD-SCID mice 6weeks following transplantation of total liver cells, sorted total livercells, or sorted R2 5E12⁺ HLA^(low) cells. The data demonstrate theengraftment and generation of functional hepatocytes from LEC.

FIGS. 7A-7F show detection of human ALB or CK19 protein in engraftedhuman fetal liver cells within the liver of a NOD-SCID mouse 6 weeksfollowing transplantation. FIGS. 10A-10F are serial sections from asingle liver. These data demonstrate the ability of LEC to generatehepatocytes. The areas where human albumin is expressed are alsopositive for CK1.9:

FIG. 8A shows the staining of human adult liver cells for the R1 and R2populations; and the staining of the staining of the R2 population for5E12, HLA. FIG. 8B shows the expression of albumin and alpha-1antitrypsin after culture in vitro.

FIGS. 9A-9H show analysis of human adult liver tissue, as described forfetal cells in FIG. 3.

FIGS. 10A to 10H show the staining of LEC in fetal and adult liver.

FIG. 11 shows the morphology of the liver engrafting cells after twoweeks in culture, in which they grow as a typical epithelial cellmonolayer.

Table 2 shows limiting dilution of human liver engrafting cells.

Table 3 shows screening for LEC by immunostaining.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Liver engrafting cells (LEC) are isolated and characterized, anddemonstrated to be progenitor cells capable of developing intohepatocytes when transplanted in vivo. The cell populations enriched forliver engrafting cells are useful in transplantation to provide arecipient with restoration of liver function; for drug screening; invitro and in vivo models of hepatic development; in vitro and in vivoscreening assays to define growth and differentiation factors, and tocharacterize genes involved in liver development and regulation; and thelike. The native cells may be used for these purposes, or they may begenetically modified to provide altered capabilities.

The ability to develop into regenerating hepatocytes can be assessed invivo, e.g. in immunodeficient animals, e.g. RAG, SCID, nude, etc., inthe FAH⁺ animals, or FAH knockout immunodeficient animals withallogeneic, syngeneic or xenogeneic donor cells, by the ability of thesedonor cells to provide functionality in this system. FAH expression is adefect of the human genetic disorder, tyrosinemia type 1. FAH functionis provided by the engrafted hepatocytes. Alternatively, in vitromethods may be used for the assessment of biological function, by thecultivation of with appropriate growth factors and/or cytokines underhepatocyte generating conditions. When grown in culture, the subjectcells grow as a monolayer, with a typical epithelial cell morphology.

The liver engrafting cells of the present invention may be enriched onthe basis of viability, forward scatter, autofluorescence, andexpression of cell surface markers. For example, after staining withpropidium iodide (PI) dead cells stain brightly because they are unableto exclude the dye. Whereas viable cells are negative to low whenstained with propidium iodide. The cells of interest are found in thePI^(low) subpopulation, between the very bright and the negative, asshown in FIG. 1. Forward scatter may also be used to gate for the cellsof interest, which have a high forward scatter; as shown in FIG. 1.

Within the population of high forward scatter, PI^(low) cells, the liverengrafting cells are positively and/or negatively selected forexpression of specific markers. By flow cytometry analysis and sortingof cell surface markers, such as those described below, viable cells canbe sorted. One such marker of interest for positive selection is the5E12 epitope. Other markers, that may be used interchangeably with 5E12for positive selection, include ep-cam, e-cadherin, and CD49f.Preferably the cells are also selected for low expression of HLA Class Iantigens, i.e. HLA-A, HLA-B and HLA-C. Other markers, that may be usedinterchangeably with HLA Class I antigens, include CD38 and CD54.Additionally, the cells may also be negatively selected, orcharacterized as negative for, expression of CD117 and/or CD14. Althoughnot usually used for selection, expression of both cytoplasmic proteinsalbumin and CK19 is characteristic of LEC.

DEFINITIONS

In the definitions of markers and cells provided below, the terms willtypically be defined in terms of human proteins, cells, and the like,where human cells are a preferred embodiment of the invention. It willbe understood by those of skill in the art that other mammals may alsobe used as a source of cells, and that selection of cells from suchnon-human species will utilize the counterpart homologous andfunctionally related markers for that species.

Liver engraftment. As used herein, the term “liver engrafting cells”refers to a progenitor cell population that, when transplanted into ananimal, gives rise to mature hepatocytes. The developmental potential ofliver progenitor cells can be assessed by functional and phenotypiccriteria. Functionally, hepatocytes are characterized by their abilityto complement FAH deficiency, and by the expression of liver specificproteins, including albumin, alpha-l-antitrypsin, alpha fetoprotein,etc. Hepatocytes are also functionally characterized by their ability tobe infected by hepatitis viruses, e.g. Hepatitis A (HAV); Hepatitis B(HBV), hepatitis C (HCV); Hepatitis D (HDV); Hepatitis E (HEV); etc. Theliver engrafting cells of the invention are also able to give rise toBEC, which can be functionally characterized by expression ofcytokeratin 19, by multicellular ductal formation and the formation ofbiliary canaliculi between individual monolayer cells.

Positive and negative staining. The subject liver engrafting cells arecharacterized: by their expression of cell surface markers. While it iscommonplace in the-art to refer to- cells as “positive” or “negative”for a particular marker, actual expression levels: are a quantitativetrait. The number of molecules on the cell surface can vary by severallogs, yet still be characterized as “positive”. It is also understood bythose of skill in the art that a cell which is negative for staining,i.e. the level of binding of a marker specific reagent is not detectablydifferent from a control, e.g. an isotype matched control; may expressminor amounts of the marker. Characterization of the level of stainingpermits subtle distinctions between cell populations.

The staining intensity of cells can be monitored by flow cytometry,where lasers detect the quantitative levels of fluorochrome (which isproportional to the amount of cell surface marker bound by specificreagents, e.g. antibodies). Flow cytometry, or FACS, can also be used toseparate cell populations based on the intensity of binding to aspecific reagent, as well as other parameters such as cell size andlight scatter. Although the absolute level of staining may differ with aparticular fluorochrome and reagent preparation, the data can benormalized to a control.

In order to normalize the distribution to a control, each cell isrecorded as a data point having a particular intensity of staining.These data points may be displayed according to a log scale, where theunit of measure is arbitrary staining intensity. In one example, thebrightest stained cells in a sample can be as much as 4 logs moreintense than unstained cells. When displayed in this manner, it is clearthat the cells falling in the highest log of staining intensity arebright, while those in the lowest intensity are negative. The “low”positively stained cells have a level of staining above the brightnessof an isotype matched control, but is not as intense as the mostbrightly staining cells normally found in the population. Low positivecells may have unique properties that differ from the negative andbrightly stained positive cells of the sample. An alternative controlmay utilize a substrate having a defined density of marker on itssurface, for example a fabricated bead or cell line, which provides thepositive control for intensity.

Sources of Progenitor Cells. Ex vivo and in vitro cell populationsuseful as a source of cells may include fresh or frozen liver cellpopulations, bile duct cell populations, or pancreatic cell populations,etc. obtained from embryonic, fetal, pediatric or adult tissue. Themethods can include further enrichment or purification procedures orsteps for cell isolation by positive selection for other cell specificmarkers. The progenitor cells may be obtained from any mammalianspecies, e.g. human, equine, bovine, porcine, canine, feline, rodent,e.g. mice, rats, hamster, primate, etc.

R2 population. Populations of cells comprising liver engraftingprogenitor cells as described above can be separated on the basis offorward scatter, autofluorescence, and viability in the presence of avital dye (such as propidium iodide, 7-MD, etc). The R2 population, asused herein, refers to a population of live, high forward scatter,autofluorescent cells, as shown in FIG. 1. After staining with the vitaldye propidium iodide (PI) the cells of interest do not stain brightly,i.e. they are (PI^(low)) This population of cells is enriched for liverengrafting progenitor cells and also contains some contaminating cells,which may be fibroblasts, endothelial cells, and the like.

5E12. The liver engrafting cells of the invention are positive forexpression of 5E12 antigen. The 5E12 monoclonal antibody was originallyraised against human neural cells. The antibody recognizes a protein ofapproximately 125 kDa. The hybridoma cell line producing the 5E12monoclonal antibody has been deposited with the American Type CultureCollection, accession number PTA-994, on Dec. 20, 1999.

Ep-cam. The liver engrafting cells of the invention are positive forexpression of ep-cam. This antigen is also known as epithelial surfaceantigen (ESA) and epithelial glycoprotein 2 (EGP-2). Ep-cam mediatesCa2+-independent homotypic cell-cell adhesions. In vivo expression ofEp-CAM is related to increased epithelial proliferation and negativelycorrelates with cell differentiation. A regulatory function of Ep-CAM inthe morphogenesis of epithelial tissue has been demonstrated for anumber of tissues. The sequence is disclosed by Szala et al. (1990)Proc. Nat. Acad. Sci. 87:3542-3546. Antibodies are commerciallyavailable, for example from BD Biosciences, Pharmingen, San Diego,Calif., catalog number 347197.

E-cadherin. The liver engrafting cells of the invention are positive forexpression of e-cadherin. E-Cadherin is a 120 kDa transmembraneglycoprotein that is localized in the adherens junctions of epithelialcells. It confers calcium-dependent cell-cell adhesion through fiveextracellular calcium-binding repeats. Expression on the cell surfaceleads to cell sorting, -hemophilic interaction specificity beingconferred by the specific extracellular regions. The intracellularregions links it with the cytoplasmic partners β-catenin or, plakoglobin(PG) and consequently to α-catenin and the actin filament network.Antibodies are commercially available, for example from BD Biosciences,Pharmingen, San Diego, Calif., catalog number 610181.

CD49f. The liver engrafting cells of the invention are positive forexpression of CD49f. Integrin alpha-6 (CD49f) is a 150 kDa transmembraneprotein, which is part of an integrin heterodimer expressedpredominantly by epithelial cells. Alpha 6 associates with integrin 131chain to form VIA-6 and with integrin (34 chain to form the laminin andkalinin receptors. CD49f is expressed mainly on T cells, monocytes,platelets, epithelial and endothelial cells, perineural cells andtrophoblasts of placenta. Its sequence may be found in Tamura et al.(1990) J. Cell Biol. 111:1593-1604. Antibodies are commerciallyavailable, for example from BD Biosciences, Pharmingen, San Diego,Calif., catalog number 557511.

HLA Class I. The liver engrafting cells of the invention are negative tolow for class I HLA expression. Examples of class I loci are HLA-A, -B,and -C. The class I MHC antigens are polymorphic 2-chain cell surfaceglycoproteins. The light chain of class I antigens isbeta-2-microglobulin. The heavy chain has a molecular weight of 44,000and is made up of 3 N-terminal extracellular domains of 90 amino acidseach, a small hydrophobic membrane-spanning segment and a smallhydrophilic intracellular C-terminal domain, see Malissen et al. (1982)Proc. Nat. Acad. Sci. 79: 893-897. Antibodies are commerciallyavailable, for example from BD Biosciences, Pharmingen, San Diego,Calif., catalog number 557349, which reacts with the human form of amonomorphic epitope of major histocompatibility class I antigens.

CD 54. The liver engrafting cells of the invention isolated from adultliver tissue are negative for expression of CD54. Cells isolated fromfetal tissue may be negative or positive for expression of CD54, but aregenerally less bright than CD54 positive cells, e.g. cells found in the5E12-population. CD54 is also known as intercellular adhesion molecule(ICAM-1), 90 (kDa). The CD54 antigen is a ligand for the leucocytefunction-associated antigen-1 (CD11 a/CD18) and influences bothLEA-1-dependent adhesion of leucocytes to endothelial cells and immunefunctions involving cell-to-cell contact: The CD54 antigen can beinducible on fibroblasts, epithelial cells, and endothelial cells. Innormal tissue, CD54 antigen density is highest in endothelium and isincreased by factors such as exposure to cytokines, inflammation, andneoplastic transformation. The nucleotide sequence of ICAM-1 is,disclosed by Simmons et al. (1988) Nature 331:624-627, 1988. Antibodiesare commercially available, for example from BD Biosciences, Pharmingen,San Diego, Calif., Cat. No. 347977.

CD117. The liver engrafting cells of the invention are negative forexpression of CD117. CD117 recognizes the receptor tyrosine kinasec-Kit. This receptor has been particularly implicated with stem cells,including hematopoietic stem cells. Multiple isoforms of c-Kit alsoexist as a result of alternate mRNA splicing, proteolytic cleavage andthe use of cryptic internal promoters in certain cell types.Structurally, c-Kit contains five immunoglobulin-like domainsextracellularly and a catalytic domain divided into two regions by a 77amino acid insert intracellularly; the sequence may be found in Yardenet. al. (1987) EMBO J. 6 (11):3341-3351. Antibodies are commerciallyavailable, for example from BD Biosciences, Pharmingen, San Diego,Calif., Cat. No. 340529.

CD14. The liver engrafting cells of the invention are negative forexpression of CD14. CD14 is a single-copy gene encoding 2 protein forms:a 50- to 55-kD glycosylphosphatidylinositol-anchored membrane protein(mCD14) and a monocyte or liver-derived soluble serum protein (sCD14)that lacks the anchor. Both molecules are critical forlipopolysaccharide (LPS)-dependent signal transduction, and sCD14confers LPS sensitivity to cells lacking mCD14. The sequence may befound in Govert et al. (1988) Science 239:497-500. Antibodies arecommercially available, for example from BD Biosciences, Pharmingen, SanDiego, Calif.

CD34. The liver engrafting cells of the invention may be negative orpositive for CD34 expression. CD34 is a monomeric cell surface antigenwith a molecular mass of approximately 110 kD that is selectivelyexpressed on human hematopoietic progenitor cells. The gene is expressedby small vessel endothelial cells in addition to hematopoieticprogenitor cells and is a single-chain 105-120 kDa heavily0-glycosylated transmembrane glycoprotein. The sequence is disclosed bySimmons et al. (1992) J. Immun. 148:267-271. Antibodies are commerciallyavailable, for example from BD Biosciences, Pharmingen, San Diego,Calif., catalog number 550760.

CD38. The liver engrafting cells of the invention may be negative orpositive for expression of CD38, but are generally less bright than CD38positive cells, e.g. cells found in the 5E12 population. CD38 is a300-amino acid type II transmembrane protein with a short N-terminalcytoplasmic tail and 4 C-terminal extracellular N-glycosylation sites.The sequence is disclosed by Jackson et al. (1990) J. Immun. 144:2811-2815. The marker is generally associated with lymphocytes,myeloblasts, and erythroblasts. Antibodies are commercially available,for example from BD Biosciences, Pharmingen, San Diego, Calif., catalognumber 347680.

Isolation of Liver Engrafting Cells

The subject liver engrafting cells are separated from a complex mixtureof cells by techniques that enrich for cells having the characteristicsas described. For example, a population of cells may be selected fromthe R2 population, for expression of one or more of 5E12, e-cadherin,ep-cam and CD49f. The cells are optionally selected for low or negativeexpression of HLA Class I antigens (herein termed HLA^(low)) CD54 andCD38 may be used interchangeably with HLA.

For isolation of cells from tissue, an appropriate solution may be usedfor dispersion or suspension. Such solution will generally be a balancedsalt solution, e.g. normal saline, PBS, Hanks balanced salt solution,etc., conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc.

The subject cells are large, blast cells, therefore an initialseparation may select for large cells by various methods known in theart, including elutriation, Ficoll-Hypaque or flow cytometry using theparameters of forward and obtuse scatter to gate for blast cells

Separation of the subject cell population will then use affinityseparation to provide a substantially pure population. Techniques foraffinity separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody or used in conjunction with amonoclonal antibody, e.g. complement and cytotoxins, and “panning” withantibody attached to a solid matrix, e.g. plate, or other convenienttechnique. Techniques providing accurate separation include fluorescenceactivated cell sorters, which can have varying degrees ofsophistication, such as multiple color channels, low angle and obtuselight scattering detecting channels, impedance channels, etc. The cellsmay be selected against dead cells by employing dyes associated withdead cells (propidium iodide, 7-AAD). Any technique may be employedwhich is not unduly detrimental to the viability of the selected cells.

The affinity reagents may be specific• receptors or ligands for the cellsurface Molecules indicated above: The details of the preparation ofantibodies and their suitability, for use as specific binding membersare well known to those skilled in the art.

Of particular interest is the use of antibodies as affinity reagents.Conveniently, these antibodies are conjugated with a label for use inseparation. Labels include magnetic beads, which allow for directseparation, biotin, which can be removed with avidin or streptavidinbound to a support, fluorochromes, which can be used with a fluorescenceactivated cell sorter, or the like, to allow for ease of separation ofthe particular cell type. Fluorochromes that find use includephycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluoresceinand Texas red. Frequently each antibody is labeled with a differentfluorochrome, to permit independent sorting for each marker.

The antibodies are added to a suspension of cells, and incubated for aperiod of time sufficient to bind the available cell surface antigens.The incubation will usually be at least about 5 minutes and usually lessthan about 30 minutes. It is desirable to have a sufficientconcentration of antibodies in the reaction mixture, such that theefficiency of the separation is not limited by lack of antibody. Theappropriate concentration is determined by titration. The medium inwhich the cells are separated will be any medium which maintains theviability of the cells. A preferred medium is phosphate buffered salinecontaining from 0.1 to 0.5% BSA. Various media are commerciallyavailable and may be used according to the nature of the cells,including Dulbeccos Modified Eagle Medium (dMEM), Hank's Basic SaltSolution (HBSS), Dulbeccos phosphate buffered saline (dPBS), RPMI,Iscoves medium, PBS with 5 mM EDTA, etc., frequently supplemented withfetal calf serum, BSA, HSA, etc.

The labeled cells are then separated as to the phenotype describedabove. The separated cells may be collected in any appropriate mediumthat maintains the viability of the cells, usually having a cushion ofserum at the bottom of the collection tube. Various media arecommercially available and may be used according to the nature of thecells, including dMEM, HBSS, dPBS, RPMI, Iscoves medium, etc.,frequently supplemented with fetal calf serum.

Compositions highly enriched for liver engrafting activity are achievedin this manner. The subject population will be at or about 50% or moreof the cell composition, and usually at or about 90% or more of the cellcomposition, and may be as much as about 95% or more of the live cellpopulation. The enriched cell population may be used immediately, or maybe frozen at liquid nitrogen temperatures and stored for long periods oftime, being thawed and capable of being reused. The cells will usuallybe stored in 10% DMSO, 50% FCS: 40% RPMI 1640 medium. Once thawed, thecells may be expanded by use of growth factors and/or stromal cells forproliferation and differentiation.

The present methods are useful in the development of an in vitro or invivo model for hepatocyte functions and are also useful inexperimentation on gene therapy and for artificial organ construction.The developing hepatocytes serve as a valuable source of novel growthfactors and pharmaceuticals and for the production of viruses orvaccines (e.g., hepatitis viruses), as well as for the study of liverparasites or of parasites having a stage of development in the liver,e.g. malarial organisms), for in vitro toxicity and metabolism testingof drugs and industrial compounds, for gene therapy experimentation(since the liver is the largest vascular organ of the body), for theconstruction of artificial transplantable livers, and for livermutagenesis and carcinogenesis studies.

Functional Assays

An assay of interest for determining the in vivo capability of hepaticprogenitor cells is an animal model of hereditary tyrosinemia type 1, asevere autosomal recessive metabolic disease which affects the liver andkidneys and which is caused by deficiency of the enzymefumarylacetoacetate hydrolase (FAH). Treatment of mice homozygous forthe FAH gene disruption (FAH⁺) with2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione (NTBC)abolishes neonatal lethality and corrects liver and kidneys functions.The animal model is described, for example, by Grompe et al. (1995)Nature Genetics 10:453-460; Overturf of al. (1996) Nat. Genet.12(3):266-73; etc.

In one embodiment of the invention, an FAH mouse is reconstituted withliver engrafting cells, which may be human progenitor cells, or mousecells comprising a detectable marker. For example, the cells may beintroduced into the mouse, which may be an irradiated mouse, and allowedto first reconstitute the liver, then NTBC is withdrawn in order toselect for hepatic reconstitution. Alternatively, NTBC may be withdrawnimmediately after introduction of the liver engrafting cells. Thereconstituted animals are useful for screening vaccines and antiviralagents against hepatic viruses, e.g. Hepatitis A, B, C, D, E; metabolicand toxicity testing of biologically active agents; and the like.

In Vitro Cell Culture

The enriched cell population may be grown in vitro under various cultureconditions. When grown in culture; the subject cells grow as amonolayer, with a typical epithelial cell morphology. Culture medium maybe liquid or semi-solid, e.g. containing agar, methylcellulose, etc: Thecell population may be conveniently suspended in an appropriate nutrientmedium, such as Iscove's modified DMEM or RPMI-1640, normallysupplemented with fetal calf serum (about 5-10%), L-glutamine, a thiol,particularly 2-mercaptoethanol, and antibiotics, e.g. penicillin andstreptomycin.

The subject cells may be grown in a co-culture with feeder layer cells.Stromal cells suitable for use as feeder layers include bone marrowstromal cells, e.g. the SYS-1 cell line, FFS-1 fibroblast cell line,etc. Other cells that can be used as a feeder layer include fibroblastsderived from human or other animal sources; fetal fibroblasts derived byprimary culture from the same species as the liver; the STO fibroblastcell line; etc. These cell layers provide non-defined components to themedium and may restrain the differentiation of the pluripotent cells.Culture in the presence of feeder layers is particularly useful forclonal culture, i.e. where a single progenitor cell is expanded to apopulation.

Functional assays may be performed using in vitro cultured cells,particularly clonogenic cultures of cells. For example, cultured cellsmay be assessed for their ability to express liver specific proteins,including albumin and alpha-1 antitrypsin. Expression may utilize anyconvenient format, including RT-PCR, ELISA for presence of the proteinin culture superannuates, etc. Cultured cells may also be assessed fortheir ability to express bile duct proteins, e.g. CK19.

The culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors. Specific growth factors that may be used inculturing the subject cells include but are not limited to hepatocytegrowth factor/scatter factor (HGF), EGF, TGFa, acidic FGF (see Block etal; J. Biol Chem, 1996 132:1133-1149). The specific culture conditionsare chosen to achieve a particular purpose, i.e. maintenance ofprogenitor cell activity, etc. In addition to, or instead of growthfactors, the subject cells may be grown in a co-culture with stromal orfeeder layer cells. Feeder layer cells suitable for use in the growth ofprogenitor cells are known in the art.

The subject co-cultured cells may be used in a variety of ways. Forexample, the nutrient medium, which is a conditioned medium, may beisolated at various stages and the components- analyzed. Separation canbe achieved with HPLC, reversed-phase-HPLC, gel electrophoresis,isoelectric focusing, dialysis, or other non-degradative techniques,which allow for separation by molecular weight, molecular volume,charge, combinations thereof, or the like. One or more of thesetechniques may be combined to enrich further for specific fractions thatpromote progenitor cell activity.

The subject cells can be expanded in culture in a stromal cell-freemedium, e.g. as described by Suzuki et al. (2000) Hepatology32:1230-1239. Such cultures preferably are grown on a substrate giving acoating of extracellular matrix components(s), e.g. laminin, Type IVcollagen, Type I collagen, fibronectin, etc. The medium generallycomprises growth factors, e.g. HGF, EGF, etc.

The liver engrafting cells may be used in conjunction with a culturesystem in the isolation and evaluation of factors associated with thedifferentiation and maturation of hepatocytes and BEC. Thus, the cellsmay be used in assays to determine the activity of media, such asconditioned media, evaluate fluids for growth factor activity,involvement with formation of specific structures, or the like. Culturesmay also be used as a means of processing drugs and other compounds, todetermine the effect of liver metabolism on an agent of interest. Forexample, the product of liver metabolism may be isolated and tested fortoxicity and efficacy.

Transplantation

Hepatic failure involves the systemic complications associated withsevere liver injury and dysfunction. It may occur in a patient withoutpre-existing liver disease or may be superimposed on chronic liverinjury. The diagnosis of acute liver failure requires the presence ofsymptoms, including jaundice and encephalopathy. Fulminant hepaticfailure impairs all liver functions, causing decreased bilirubinmetabolism, decreased clearance of ammonia and gut-derived proteins, anddecreased clotting factor production. It may also cause kidney failure,shock, and sepsis. Without a liver transplant, more than 50% of patientswill die, usually from a combination of the above conditions. Mortalityexceeds 50%, even in the best circumstances. Management involves generalsupportive measures until the liver can regenerate and resume function.In acute liver failure without pre-existing disease, liver transplantcan be life-saving.

The subject cells may be used for reconstitution of liver function in arecipient. Allogeneic cells may be used for progenitor cell isolationand subsequent transplantation. Most of the clinical manifestations ofliver dysfunction arise from cell damage and impairment of the normalliver capacities. For example, viral hepatitis causes damage and deathof hepatocytes. In this case, manifestations may include increasedbleeding, jaundice, and increased levels of circulating hepatocyteenzymes. Where the liver dysfunction arises from conditions such astumors, the subject cells can be isolated from the autologous livertissue, and used to regenerate liver function after treatment.

Liver disease has numerous causes, ranging from microbial infections andneoplasms (tumors) to metabolic and circulatory problems. Hepatitisinvolves inflammation and damage to the hepatocytes. This type of insultmay result from infectious agents, toxins, or immunologic attack.However, the most common cause of hepatitis is viral infection. Threemajor viruses cause hepatitis in the United States: hepatitis viruses A,B, and C. Together, they infect nearly 500,000 people in the UnitedStates every year. In addition, bacteria, fungi, and protozoa can infectthe liver, and the liver is almost inevitably involved to some extent inall blood-borne infections.

Numerous medications can damage the liver, ranging from mild,asymptomatic alteration in liver chemistries to hepatic failure anddeath. Liver toxicity may or may not be dose-related.Tylenol.(Acetominophen) is an hepatotoxic drug; Dilantin (ananti-convulsant) and isoniazid (an anti-tuberculosis agent) are examplesof drugs that can cause “viral-like” hepatitis. Both environmental andindustrial toxins can cause a wide variety of changes in the liver.Hepatic damage is not necessarily dose-dependent and can range frommild, asymptomatic inflammation to fulminant failure or progressivefibrosis and cirrhosis.

Problems with metabolic processes in the liver can be either congenitalor acquired. Some of these disorders, such as Wilson's disease andhemochromatosis, can present as hepatitis or cirrhosis. Wilson's diseaseis a rare inherited condition characterized by an inability to excretecopper into bile, resulting in the toxic accumulation of copper in theliver and nervous system. Hemochromatosis is an iron overload syndromecausing iron deposits and consequent damage to various organs, includingthe liver, heart, pancreas, and pituitary gland. The disease may be dueto an inherited increase in gut absorption of iron or to multiple bloodtransfusions, since iron is normally found in circulating red bloodcells.

The liver may be affected by numerous conditions, particularlyautoimmune disorders, in which the immune system attacks the body's ownnormal tissues. Some examples include rheumatic diseases, such assystemic lupus erythematosus and rheumatoid arthritis, and inflammatorybowel diseases, such as ulcerative colitis and Crohn's disease.

Genes may be introduced into the cells prior to culture ortransplantation for a variety, of purposes e.g. prevent or reducesusceptibility to infection, replace genes having a loss of functionmutation, etc. Alternatively, vectors are introduced that expressantisense mRNA or ribozymes, thereby blocking expression of an undesiredgene. Other methods of gene therapy are the introduction of drugresistance genes to enable normal progenitor cells to have an advantageand be subject to selective pressure, for example the multiple drugresistance gene (MDR), or anti-apoptosis genes, such as bcl-2. Varioustechniques known in the art may be used to transfect the target cells,e.g. electroporation, calcium precipitated DNA, fusion, transfection,lipofection and the like. The particular manner in which the DNA isintroduced is not critical to the practice of the invention.

Many vectors useful for transferring exogenous genes into mammaliancells are available. The vectors may be episomal, e.g. plasmids, virusderived vectors such cytomegalovirus, adenovirus, etc., or may beintegrated into the target cell genome, through homologous recombinationor random integration, e.g. retrovirus derived vectors such MMLV, HIV-1,ALV, etc. For examples of progenitor and stem cell genetic alteration,see Svendsen et al. (1999) Trends Neurosci. 22(8):357-64; Krawetz et al.(1999) Gene 234(1):1-9; Pellegrini et al. Med Biol Eng Comput.36(6):778-90; and Alison (1998) Curr Opin Cell Biol. 10(6):710-5.

Alternatively, the liver progenitors can be immortalized-disimmortalized(for example, see Kobayashi et al. (2000) Science 287:1258-1262. In sucha procedure, an immortalizing genetic sequence, e.g. an oncogene, isintroduced into the cell, in such a manner hat it can be readilyremoved, for example with a site specific recombinase such as thecre-lox system.

To prove that one has genetically modified progenitor cells, varioustechniques may be employed. The genome of the cells may be digested withrestriction enzymes and used with or without DNA amplification. Thepolymerise chain reaction; gel electrophoresis; restriction analysis;Southern, Northern, and Western blots; sequencing; or the like, may allbe employed. The cells may be grown under various conditions to ensurethat the cells are capable of differentiation while maintaining theability to express the introduced DNA. Various tests in vitro and invivo may be employed to ensure that the pluripotent capability of thecells has been maintained.

The cells may be administered in any physiologically acceptable medium,normally intravascularly, including intravenous, e.g. through thehepatic portal vein; intrasplenic, etc. although they may also beintroduced into other convenient sites, where the cells may find anappropriate site for regeneration and differentiation. Usually, at least1×10³/Kg cells will be administered, more usually at least about1×10⁴/Kg, preferably 1×10⁶/Kg or more. The cells maybe introduced byinjection, catheter, or the like

The subject cells find use in as cultured cells, and for the generationof hepatocytes for bioartificial liver bioreactors, in which thehepatocytes are separated by a membrane or other physical barrier fromthe perfusate stream. Four devices (Circe Biomedical HepatAssist®,Vitagen ELAD™, Gerlach BELS, and Excorp Medical BLSS) that utilizehepatocytes cultured in hollow-fiber membrane are currently in clinicalevaluation. While the development of bioartificial liver assist devices(BLADs) for the treatment of acute liver failure, either fulminant oracute decompensation on chronic liver failure, is of great interest, ithas been difficult to accomplish, in part because hepatocytes areextremely difficult to maintain in culture. By culturing the subjectliver engrafting cells, a constant supply of hepatocytes is provided forsuch devices.

Bioartificial liver bioreactors provides one or more of the functions:oxidative detoxification (primarily through the cytochrome P450 enzymesystem); biotransformation (e.g., urea synthesis, gluconuridation, andsulfation); excretion (through the bile system); protein andmacromolecule synthesis; intermediate metabolism (gluconeogenesis, fattyacid, and amino acid); and immune and hormonal system modulation.

Current BLADs in clinical evaluation are based on the use ofhollow-fiber cartridges housing hepatocytes cultured in the extraluminalspace of the hollow fibers. Perfused through the luminal space of thehollow fiber cartridge are whole blood, or a plasma stream. Anoxygenator may be placed before the bioreactors to raise tile availableoxygen levels in the perfusing stream, and columns or filters used toreduce toxins prior to reaching the hepatocytes.

Other devices may perfuse plasma in an axial flow path over and/orthrough a nonwoven polyester fabric; through channels cored in an highlyporous polyurethane foam structure seeded with hepatocytes; throughmicroporous polysulfone hollow-fiber membranes; microporous polyvinylformal resin material; and the like. The progenitor cells, and orprogeny hepatocytes may be encapsulated.

Expression Assays

Of particular interest is the examination of gene expression in liverengrafting cells. The expressed set of genes may be compared with avariety of cells of interest, e.g. adult hepatic progenitor cells, stemcells, hematopoietic cells, etc., as known in the art. For example, onecould perform experiments to determine the genes that are regulatedduring development.

Any suitable qualitative or quantitative methods known in the art fordetecting specific mRNAs can be used. mRNA can be detected by, forexample, hybridization to a microarray, in situ, hybridization in tissuesections, by reverse transcriptase-PCR; or in Northem, blotscontaining-poly A⁺ mRNA. One of skill in the art can readily use thesemethods to determine differences in the size or amount of mRNAtranscripts between two samples. For example, the level of particularmRNAs in progenitor cells is compared with the expression of the mRNAsin a reference sample, e.g. hepatocytes, or other differentiated cells.

Any suitable method for detecting and comparing mRNA expression levelsin a sample can be used in connection with the methods of the invention.For example, mRNA expression levels in a sample can be determined bygeneration of a library of expressed sequence tags (ESTs) from a sample.Enumeration of the relative representation of ESTs within the librarycan be used to approximate the relative representation of a genetranscript within the starting sample. The results of EST analysis of atest sample can then be compared to EST analysis of a reference sampleto determine the relative expression levels of a selectedpolynucleotide, particularly a polynucleotide corresponding to one ormore of the differentially expressed genes described herein.

Alternatively, gene expression in a test sample can be performed usingserial analysis of gene expression (SAGE) methodology (Velculescu etal., Science (1995) 270:484). SAGE involves the isolation of shortunique sequence tags from a specific location within each transcript.The sequence tags are concatenated, cloned, and sequenced. The frequencyof particular transcripts within the starting sample is reflected by thenumber of times the associated sequence tag is encountered with thesequence population.

Gene expression in a test sample can also be analyzed using differentialdisplay (DD) methodology. In DD, fragments defined by specificpolynucleotide sequences (or restriction enzyme sites) are used asunique identifiers of genes, coupled with information about fragmentlength or fragment location within the expressed gene. The relativerepresentation of an expressed gene with in a sample can then beestimated based on the relative representation of the fragmentassociated with that gene within the pool of all possible fragments.Methods and compositions for carrying out DD are well known in the art,see, e.g., U.S. Pat. Nos. 5,776,683; and 5,807,680.

Alternatively, gene expression in a sample using hybridization analysis,which is based on the specificity of nucleotide interactions.Oligonucleotides or cDNA can be used to selectively identify or captureDNA or RNA of specific sequence composition, and the amount of RNA orcDNA hybridized to a known capture sequence determined qualitatively orquantitatively, to provide information about the relative representationof a particular message within the pool of cellular messages in asample. Hybridization analysis can be designed to allow for concurrentscreening of the relative expression of hundreds to thousands of genesby using, for example, array-based technologies having high densityformats, including filters, microscope slides, or microchips, orsolution-based technologies that use spectroscopic analysis (e.g., massspectrometry). One exemplary use of arrays in the diagnostic methods ofthe invention is described below in more detail.

Hybridization to arrays may be performed, where the arrays can beproduced according to any suitable methods known in the art. Forexample, methods of producing large arrays of oligonucleotides aredescribed in U.S. Pat. Nos. 5,134,854, and 5,445,934 usinglight-directed synthesis techniques. Using a computer controlled system,a heterogeneous array of monomers is converted, through simultaneouscoupling at a number of reaction sites, into a heterogeneous array ofpolymers. Alternatively, microarrays are generated by deposition ofpre-synthesized oligonucleotides onto a solid substrate, for example asdescribed in PCT published application no. WO 95/35505.

Methods for collection of data from hybridization of samples with anarrays are also well known in the art. For example, the polynucleotidesof the cell samples can be generated using a detectable fluorescentlabel, and hybridization of the polynucleotides in the samples detectedby scanning the microarrays for the presence of the detectable label.Methods and devices for detecting fluorescently marked targets ondevices are known in the art. Generally, such detection devices includea microscope and light source for directing light at a substrate. Aphoton counter detects fluorescence from the substrate, while an x-ytranslation stage varies the location of the substrate. A confocaldetection device that can be used in the subject methods is described inU.S. Pat. No. 5,631,734. A scanning laser microscope is described inShalon et al., Genome Res. (1996) 6:639. A scan, using the appropriateexcitation line, is performed for each fluorophore used. The digitalimages generated from the scan are then combined for subsequentanalysis. For any particular array element, the ratio of the fluorescentsignal from one sample is compared to the fluorescent signal fromanother sample, and the relative signal intensity determined.

Methods for analyzing the data collected from hybridization to arraysare well known in the art. For example, where detection of hybridizationinvolves a fluorescent label, data analysis can include the steps ofdetermining fluorescent intensity as a function of substrate positionfrom the data collected, removing outliers, i.e. data deviating from apredetermined statistical distribution, and calculating the relativebinding affinity of the targets from the remaining data. The resultingdata can be displayed as an image with the intensity in each regionvarying according to the binding affinity between targets and probes.

Pattern matching can be performed manually; or can be performed using acomputer program. Methods for preparation of substrate matrices (e.g.,arrays), design of oligonucleotides for use with such matrices, labelingof probes, hybridization conditions, scanning of hybridized matrices,and analysis of patterns generated, including comparison analysis, aredescribed in, for example, U.S. Pat. No. 5,800,992.

In another screening method, the test sample is assayed at the proteinlevel. Diagnosis can be accomplished using any of a number of methods todetermine the absence or presence or altered amounts of a differentiallyexpressed polypeptide in the test sample. For example, detection canutilize staining of cells or histological sections (e.g., from a biopsysample) with labeled antibodies, performed in accordance withconventional methods. Cells can be permeabilized to stain cytoplasmicmolecules. In general, antibodies that specifically bind adifferentially expressed polypeptide of the invention are added to asample, and incubated for a period of time sufficient to allow bindingto the epitope, usually at least about 10 minutes. The antibody can bedetectably labeled for direct detection (e.g., using radioisotopes,enzymes, fluorescers, chemiluminescers, and the like), or can be used inconjunction with a second stage antibody or reagent to detect binding(e.g., biotin with horseradish peroxidase-conjugated avidin, a secondaryantibody conjugated to a fluorescent compound, e.g. fluorescein,rhodamine, Texas red, etc.). The absence or presence of antibody bindingcan be determined by various methods, including flow cytometry ofdissociated cells, microscopy, radiography, scintillation counting, etc.Any suitable alternative methods of qualitative or quantitativedetection of levels or amounts of differentially expressed polypeptidecan be used, for example ELISA, western blot, immunoprecipitation,radioimmunoassay, etc.

Screening Assays

The subject cells are useful for in vitro assays and screening to detectagents that affect liver engrafting cells and hepatocytes generated fromthe liver engrafting cells. A wide variety of assays may be used forthis purpose, including toxicology testing, immunoassays for proteinbinding; determination of cell growth, differentiation and functionalactivity; production of hormones; and the like.

In screening assays for biologically active agents, viruses, etc. thesubject cells, usually a culture comprising the subject cells, iscontacted with the agent of interest, and the effect of the agentassessed by monitoring output parameters, such as expression of markers,cell viability, and the like. The cells may be freshly isolated,cultured, genetically altered as described above; or the like. The cellsmay be environmentally induced variants of clonal cultures: e.g. splitinto independent cultures and grown under distinct conditions, forexample with or without virus; in the presence or absence of othercytokines or combinations thereof. The manner in which cells respond toan agent, particularly a pharmacologic agent, including the timing ofresponses, is an important reflection of the physiologic state of thecell.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

Agents of interest for screening include known and unknown compoundsthat encompass numerous chemical classes, primarily organic molecules,which may include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the invention is to evaluatecandidate drugs, including toxicity testing, to test the effect ofhepatic viruses, e.g. Hepatitis A, B, C, D, E viruses; antiviral agents;and the like.

In addition to complex biological agents, such as viruses, candidateagents include organic molecules comprising functional groups necessaryfor structural interactions, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, frequently at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules, including peptides, polynucleotides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,hormones or hormone antagonists; etc. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Water, Salts and Ions; DrugsAffecting Renal Function and Electrolyte Metabolism; Drugs AffectingGastrointestinal Function; Chemotherapy of Microbial Diseases;Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Formingorgans; Hormones and Hormone Antagonists; Vitamins, Dermatology; andToxicology, all incorporated herein by reference. Also included aretoxins, and biological and chemical warfare agents, for example seeSomani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, NewYork, 1992).

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

The term samples also includes the fluids described above to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1 μlto 1 ml of a biological sample is sufficient.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cell samples, usually inconjunction with cells lacking the agent. The change in parameters inresponse to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

Various methods can be utilized for quantifying the presence of theselected markers. For measuring the amount of a molecule that ispresent, a convenient method is to label a molecule with a detectablemoiety, which may be fluorescent, luminescent, radioactive,enzymatically-active, etc., particularly a molecule specific for bindingto the parameter with high affinity Fluorescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g. by expressing them asgreen fluorescent protein chimeras inside cells (for a review see Joneset el. (1999) Trends Biotechnol. 17(12):477-81). Thus, antibodies can begenetically modified to provide a fluorescent dye as part of theirstructure. Depending upon the label chosen, parameters may be measuredusing other than fluorescent labels, using such immunoassay techniquesas radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.The quantitation of nucleic acids, especially messenger RNAs, is also ofinterest as a parameter. These can be measured by hybridizationtechniques that depend on the sequence of nucleic acid nucleotides.Techniques include polymerase chain reaction methods as well as genearray techniques. See Current Protocols in Molecular Biology, Ausubel etal., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999)Biotechniques 26(1):112-225; Kawamoto et al. (1999) Genome Res9(12):1305-12; and Chen et al. (1998) Genomics 51(3):313-24, forexamples.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as an:admission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

EXAMPLE 1 Flow Cytometric Sorting of Human Liver Cells

Dual laser flow cytometric analyses and sorting of human liver cellsfrom fetal and adult tissue were performed on a Becton DickinsonFACSVantage SE. An Argon Ion laser and a Helium Neon laser were utilizedas the primary and secondary excitation sources emitting 150 mW at the488 nm wavelength and 30 mW of 633 nm wavelength, respectively. Lightscattered at forward and orthogonal angles was amplified linearly andmeasured through 48 nm bandpass filters, employing a 0.6 OD neutraldensity filter in front of the forward scatter detector in order toattenuate high level forward angle scatter signals resulting frompopulations of larger sized cells. in this configuration there issufficient dynamic range on the forward scatter axis to capture andscale forward angle scatter signals, resulting from the diverse range ofcell sizes found in liver tissue, within a single linear decade. Typicalforward scatter amplifier gain settings range from 8 to 16. FITC, PE,and PI fluorochromes were all excited at 488 nm and fluorescenceemissions were measured using 530/30, 585/42, 610/20 nm bandpassfilters, respectively. APC and APC-Cy7 fluorochromes were excited at 633nm and fluorescence emissions were measured using 660/20 bandpass and750 longpass filters, respectively. All immunofluorescence measurementswere amplified logarithmically.

The voltage settings for each fluorescent channel are calibrated usingthe Spherotech RFP 30=5 reference particle. Following calibration,compensation settings are derived empirically using single colorcontrols.

Configuration of the fluidics setup on the flow cytometer is optimizedfor the unique size distribution and characteristics of cells found inliver derived tissue. A custom fabricated nozzle tip with an orificediameter of 130 pm and a sheath pressure setting of 10 psi are used. Thetemperature of the sheath reservoir, the sample holder, and the receipttube holder are all maintained at 4° C. by a refrigerated recirculator.

Clustered subpopulations of liver derived cells can be identified byadding PI to the cell suspension, subjecting the cells to flowcytometry, and analyzing the data as a plot of forward scatter versus PIfluorescence. Three distinct populations can be resolved according tothe following attributes; a small cell cluster displaying low forwardscatter, low level fluorescence (R1); a large cell cluster displayinghigh forward scatter and mid-scale fluorescence (R2), and dead cellscomprising the third distinct cluster with a continuum of low to midlevel forward scatter signal and very high level fluorescence. Resultsare shown in FIG. 1. The R2 subpopulation is demonstrated to beautofluorescent by performing the analysis described above in theabsence of PI. Two distinct populations can be resolved according to thefollowing attributes; a small cell cluster displaying low forwardscatter, low level fluorescence and a large cell cluster displaying highforward scatter and mid-scale fluorescence.

Aggregates are discriminated, using pulse processing, by plottingforward scatter peak height against width, which forms the basis of thethird region. A sort gate is defined as the intersection of these threeregions. Primary enrichment of the target population is achieved in thefirst round of sorting. The enriched product may then be re-sorted torelative purity. Product purity is always verified by re-analysis.

EXAMPLE 2 Isolation of a Liver Progenitor Cell

Liver cells previously frozen or freshly isolated liver cells werestained with 5E12. The cells were enriched for 5E12 by MACS column usinga 5E12 Ab, then 5E12+ cells were sorted. Alternatively, 5E12+ cells weresorted directly after staining. For the sorting; cells are separatedbetween RI and R2 gates. R2/5E12⁺ cells represents the majority of liverengrafting cells in an in vitro or in vivo assay. The cells werecharacterized by expression of ck19 and albumin in the same cell (shownin FIGS. 2A and 2B). In addition to 5E12, selection for lower levels ofHLA-Class I expression enriched for liver engraftment, using antibodyreactive with human HLA-Class I A,B,C:(W6-32 antibody). When gating onthe R2 cells, three distinct clustered subpopulations of cells can beresolved by analysis of 5E12 fluorescence versus HLA-Class Ifluorescence as two-dimensional density or contour plots (FIG. 1). Onesubpopulation displays negative staining for both 5E12 and HLA-Class I(5E12 HLA); a second subpopulation displays a lower relative level of5E12 fluorescence and higher levels of HLA-Class I fluorescence (5E1.2HLA⁺); and a third subpopulation displays higher level 5E12 fluorescenceand lower level HLA-Class I fluorescence (5E12+HLA^(low)).

While an analysis of a single stain, i.e. one color, does notnecessarily provide a clear population distinction, it is clear from theFigures that in a two color plot the cells fall into distinctsubpopulations.

Results

A method is provided to enrich for liver progenitor colonies by sortingfor expression of cell surface antigens. From the results of aclonogenic assay of sorted human fetal liver cells: cells were sorted byviability (PI), size (FS), and autofluorescence and were then furtherseparated by surface antibodies, and plated on FFS- or BMS-6. Theproliferative capacity of the hepatic colonies generated after sorting(ckl9/albumin or ck19 only) were compared after 2 weeks in culture. OnlyR2 gated cells (FS⁺PI^(low)) generated colonies.

The 5E12 antibody enriches for human fetal and adult liver progenitorcells. FIG. 1 illustrates the staining of human fetal liver cells (16g.w.) with 5E12 monoclonal antibodies. Cells included in the R2 gatewere stained with 5E12 or isotype matched control mAb.

Table 1 and Table 2 show the results of a limiting dilution analysis ofsorted 5E12 liver cells. Human fetal liver cells were enriched for 5E12positive cells using MACS columns. L R2, 5E12 positive cells were sortedin 96 well plates on BMS-6 stroma from 1 to 500 cells per well. Humanalbumin expression was monitored by ELISA, in order to detect coloniesof progenitor cells in the culture wells. TABLE 1 Limiting dilutionanalysis of sorted 5E12 positive cells. The analysis was done using ACDUsorted 5E12+ liver cells and 8 dilutions points (1, 5, 10, 25, 50, 125,250, 500 cells). Albumin positive wells were detected by ELISA at day 7,day 14, day 21 and day 28. Day R2/5E 12 D7 1/39 D14 1/50 D2 T 1/79 D28 1/147

EXAMPLE 3 Characterization of Liver Engrafting Cell Phenotype

Human fetal liver cells and human adult liver cells were obtainedmaintained at 4° C. To make a cell suspension the tissue was minced,resuspended in Ca++ free buffered saline, and digested with collagenasein the presence of hyaluronidase for 30 min at 37° C. Optionally thecell suspensions were additionally digested with trypsin/EDTA for 20 minat 37° C. The cell suspension was filtered through 70 pm nylon filterand resuspended in IMDM containing 2% FBS, 2 mM EDTA. To an aliquot ofcells was added a combination of two or more antibodies, previouslytitrated, as shown in FIGS. 3 and 4. Isotype matched controls wereutilized for all stainings. The cells were analyzed by flow cytometry asdescribed in Examples 1 and 2.

The data show (FIG. 3A) that CD14 does not stain the LEC cells (R2,5E12⁺ HLA^(low)). CD54, CD38 and CD34 positively stain the LEC (FIGS.3B-3D). FIGS. 4A, 4D, 4G show the typical staining pattern for LEC.FIGS. 4B, 4E and 4H show that E-Cadherin, EpCam and CD49f have astaining profile similar to that of 5E12 on the R2 population. FIGS. 4C,4F and 41 show that the LEC populations selected E-Cadherin, EpCam orCD49f are uniformly 5E12 positive. These data demonstrate that 5E12,E-cadherin, EpCam and CD49f can be used interchangeably in the selectionof LEC. Similar data were obtained with adult liver tissue (shown inFIG. 9). FIG. 10 summarizes these data.

EXAMPLE 4 In Vitro Assay for Human Liver Cells

Liver cells, including liver progenitor cells, are shown to survive andproliferate on stromal cells used as feeders. The in vitro culture ofthese cells permits isolation and characterization of progenitor cellsfrom liver. Two different feeder stromal cell lines were used asfeeders, BMS-6, a bone marrow stromal line and FFS-1, a fetalfibroblast. This assay is based on a feeder cell dependent co-culturesystem and the nature of a liver progenitor cell which should be ahighly proliferative cell with liver engrafting capability.

Materials and Methods

Feeder layer Preparation and Culture Conditions: FFS-1 murine fibroblastcells (derived from STO) were mitomycin treated (10 μg/ml, Sigma, StLouis, Mo.) for 5 hours and plated at 5×10⁴ cells/cm². BMS-6 murine bonemarrow stroma were plated at 1.6×10⁴/cm. The feeder layers were culturedin 1:1 mixture of Dulbecco's modified Eagle's medium and Medium-199 with10% FCS.

Isolation of Enriched Liver Cell Fractions using Flow Cytometry: Livercell preparations were prepared by typical tissue digestion proceduresand single cell suspensions and analyzed by multi-parameter flowcytometry. Isolation of specific liver cell subpopulations wasaccomplished using a fluorescence activated cell sorter (FACSTM)manufactured by Becton Dickinson Immunocytometry Systems. Specifically,the FACSVantage SE is configured with argon, krypton, and Helium-Neonion lasers, which deliver three spatially separate excitation sources.This setup allows us to employ a wide variety of commercially availablefluorescent probes for the analyses of discrete cellular features.Specialized subsystems built into this instrument permit the indexeddeposition of single cells directly into individual wells of tissueculture plates previously cultured with feeder layer cells. Computerassisted high-speed data acquisition systems allow the collection of upto nine independent data parameters from each single cell. The abilityto collect listmode data files comprised of more than one million eventsfacilitates the discrimination of very low frequency subpopulations.Data parameters were collected in the list mode data file and wereanalyzed by the software program Flowjo (www.Treestar.com). Purepopulations of liver cells were sorted directly into individual wells of96-well or 24-well plates previously cultured with feeder layer cells.

Protocol for freezing cells: Resuspend liver cells in IMDM+10% FCS with40% FCS for 5 minutes on ice then mix 1:1 with IMDM+10% FCS with 15%DMSO. Freeze cells at −800 C than liquid nitrogen.

Protocol for hepatitis infection: Incubate liver cells with human serumcontaining hepatitis for an hour on ice, then plate the cells on stromaand culture the cells for 2 weeks.

Results

Liver cell populations were separated into R2/5E12+ versus R2/5E12− byMACS or sorting or both. The cells were then cultured for 2 weeks andthen assessed the CFC-LBC (colony forming cell-liver bipotent colony) bythe expression of Albumin and ck19 in the forming colonies, shown inFIG. 8. The hepatic colony frequency was calculated: by limitingdilution using 3 different cell concentrations. The data is shown inTable 3, and FIG. 8 (human adult cells).

The limiting dilution assays can utilize a combination of cell sortingand ELISA to determine limiting dilutions. For example, cells weresorted according to R2 gates, expression of 5E12, and expression of HLAantigens. The sorted cells were diluted into 96 well plates as describedabove, and cultured in vitro for 14 days, then analyzed by ELISA forexpression of alpha-fetoprotein, albumin, and alpha-1 antitrypsin.

EXAMPLE 5 In Vitro Model of Hepatitis Infection

A method is provided for the in vitro infection of hepatic colonies byhepatitis viruses. Fetal liver cells (18 g.w.) containing liverengrafting progenitor cells were isolated and infected with HepatitisVirus D (HDV) and cultured for 2 weeks. Cells were cultured for twoweeks, fixed and stained for albumin (APC-blue), Cytokeratin 19(FITC-green) and HDV (PE-red) to identify hepatic progenitors infectedwith hepatitis D virus. Nuclei were counterstained with Hoechst:

Fetal liver cells were incubated with serum from patients infected withHDV virus. 1 hour later, the sample was put on stroma layer and left for2 weeks. The culture was fixed and stained with hepatic and anti-HDVmarkers. These methods of culture support the growth of cells that areinfectable with hepatitis viruses.

EXAMPLE 6 Ex vivo Expansion of Human Liver Engrafting Cell Populations

Human liver engrafting cells, which had been enriched for hepatocyteprogenitors bearing the HLA^(low) 5E12+ phenotype, were plated on or inan extracellular matrix (ECM) that provides for cellular attachment,adhesion, and proliferation. The cells were cultured in a suitable basalmedium in combination with the matrix component laminin, in the presenceof Liver engrafting cell (LEC) medium. The cell morphology is shown inFIG. 11.

Liver engrafting cell (LEC) Medium: DMEM/F12 (50:50) with L-glutamine;10% fetal bovine serum; dexamethasone (10-7M); nicotinamide (10 mM);Beta-mercaptoethanol (0.05 mM); Penicillin/streptomycin (1×);Recombinant human hepatocyte growth factor (40 ng/mL); Recombinant humanepidermal growth factor (20 ng/mL).

Throughout the course of ex vivo expansion, the clonogenic potential ofexpanded cells was assessed in a stromal coculture assay as describedabove for uncultured liver cells. The secreted hepatic proteins albumin,alpha-l-antitrypsin, or alpha-fetoprotein, were monitored by ELISAassays of culture supernatants from proliferating cells on ECM plus LECmedium or in stromal coculture.

Engraftment potential of cells expanded by culture on ECM plus LECmedium can be assessed by transplantation in various suitable animalmodels, including the NOD-SCID mouse or the NOD-SCID/FAH mouse. Anadditional approach is to induce differentiation of expanded cells as ameans to promote improved liver engraftment or long-term hepatocytefunction. Cells expanded by culture on ECM plus LEC medium may beexposed to additional growth factors, cytokines, or differentiationagents, to promote a differentiation state of a mature hepatocyte. Theimpact of such treatments on the engraftment potential of the expandedand differentiated cells can be evaluated by transplantation in animalmodels as described above.

EXAMPLE 7 Transplantation of Human Liver Engrafting Cell Populations

The engraftment and hepatocyte differentiation potential of human livercells was assessed by transplantation into the NOD-SCID mouse. Briefly,human liver cells were resuspended in a injection buffer (50% MatrigelBD Biosciences #356234, 50% DMEM) and placed on ice until injection. Upto 20 microliters of cells in injection buffer were injected into thelivers of 0-48 hours old newborn NOD-SCID mice. Serum from the injectedmice was analyzed by ELISA 5-6 weeks after transplantation for thepresence human liver-specific proteins (albumin, alpha-l-antitrypsin, oralpha-fetoprotein). In FIG. 6, circulating human alpha-1-antitrypsin(AAT) and albumin (ALB) protein was detected from serum of NOD-SCID mice6 weeks following transplantation of total liver cells, sorted totalliver cells, or sorted R2 5E12⁺ HLA^(low) cells. The levels of AAT orALB in mice engrafted with 10,000 sorted R2 5E12⁺ HLA^(low) cells wasgreater than or equal to that in mice engrafted with 75,000 unsortedtotal liver cells, or 10,000 or 40,000 sorted total liver cells. Thehuman AAT or ALB are repeatedly detectable in mice transplanted withsorted R2 5E12⁺ HLA^(low) cells for up to 5 months, indicating a durableengraftment and sustained hepatic differentiation. FIG. 7 showsdetection of human ALB or CK19 protein in engrafted human fetal livercells within the liver of a representative NOD-SCID mouse 6 weeksfollowing transplantation. The serum levels of human AAT and ALBdetected by ELISA analysis at the time of sacrifice are shown in thebottom panels.

1. A composition of human liver engrafting cells isolated from livertissue; wherein the cells in said composition are MHC class I^(negative/low); and positive for a marker selected from the groupconsisting of 5E12, Ep-Cam, CD49f, and E-Cadherin; and within saidcomposition are cells that give rise to mature hepatocytes.
 2. Thecomposition of liver engrafting cells according to claim 1, wherein saidcells are negative for CD117 and CD14.
 3. The composition of liverengrafting cells according to claim 1, wherein said cells aregenetically modified to comprise an exogenous DNA vector.
 4. A methodfor providing functional hepatocytes and/or biliary cells to a hostanimal, the method comprising: introducing into said host animal a cellpopulation comprising a substantially pure composition of mammalianliver engrafting cells, wherein at least 80% of the cells in saidcomposition are characterized as R2, and positive for a marker selectedfrom the group consisting of 5E12, Ep-Cam; CD49f; and E-Cadherin; andHLA ^(low).
 5. The method according to claim 4, wherein said cells arenegative for CD117 and CD14.
 6. The method according to claim 4, whereinsaid cells are human cells.
 7. The method according to claim 4, whereinsaid functional hepatocytes secrete albumin or alpha 1 antitrypsin. 8.The method according to claim 4, wherein said functional hepatocytessecrete alpha 1 antitrypsin.
 9. The method according to claim 4, whereinsaid functional hepatocytes secrete albumin.
 10. The composition ofliver engrafting cells according to claim 2, wherein said compositioncomprises cells that express both CK19 and albumin.
 11. A composition ofhuman liver engrafting cells isolated from liver tissue, wherein thecells in said composition are MHC class I ^(negative/low); positive fora marker selected from the group consisting of 5E12, Ep-Cam, CD49f, andE-Cadherin; negative for CD14 and CD 117; and comprises cells thatexpress both CK19 and albumin; and within said composition are cellsthat give rise to mature hepatocytes.
 12. A method of screening forgenetic sequences specifically expressed in mammalian liver engraftingcells, the method comprising: isolating RNA from a cell compositionaccording to claim 1 or claim 11, generating a probe from said RNA,screening a population of nucleic acids for hybridization to said probe.13. The method of claim 12, further comprising a comparison of thehybridization obtained between said liver engrafting cells, and a seconddistinct cell population.
 14. The method of claim 13, wherein saidpopulation of nucleic acids is represented in an array.
 15. A method ofscreening for agents that affect viability, growth, metabolic functionor differentiation of mammalian liver engrafting cells, the methodcomprising: contacting a cell composition according to claim 1, or claim11, and determining the effect of said agent on the viability, growth,metabolic function or differentiation of said liver engrafting cells.16. The method according to claim 15, wherein said agent is a drugsuspected of toxicity on human hepatocytes.
 17. The method according toclaim 15, wherein said agent is a human hepatitis virus.
 18. The methodaccording to claim 15, wherein said agent is a human hepatitis virusvaccine.
 19. The method according to claim 15, wherein said agent is ananti-viral agent.